Fig. 3

USP5 is a bona fide deubiquitinase that targets PFKP protein for deubiquitination and stabilization in TNBC. a Co-IP analysis of PFKP protein ubiquitination in MCF-10A and MDA-MB-231 cells. b MCF-10A and MDA-MB-231 cells were treated with CHX for the indicated time points and analyzed by Western blot. c Quantification of PFKP protein levels normalized to β-actin in time after the addition of CHX. d The diagram showing three deubiquitinases (USP5, USP14, and OTUB1) and three E3 ubiquitin ligases (HUWE1, TRIM21, and TRIM25) could interact with PFKP identified by a combination of Co-IP and MS. e The correlation between USP5 protein expression and PFKP protein expression in TNBC was analyzed by LinkedOmics platform. f The USP5 and PFKP protein expression in control and USP5 knockdown MDA-MB-231 cells were analyzed by Western blot. g Co-IP analysis of PFKP protein ubiquitination in control and USP5 knockdown MDA-MB-231 cells. h–k Control and USP5 knockdown MDA-MB-231 cells were treated with CHX for the indicated time points and analyzed by Western blot h, j, quantification of PFKP protein levels normalized to β-actin in time after the addition of CHX (i, k). l The binding of USP5 to PFKP was detected by Co-IP assay in MDA-MB-231 cells. m Immunofluorescence co-localization of USP5 and PFKP in MDA-MB-231 cells. n In vitro binding assays of purified GST-PFKP and His-USP5 proteins